Plague is a zoonosis that is present in wild rodent populations worldwide and is transmitted primarily by fleas. Yersinia pestis, the plague bacillus, is unique among the enteric group of gram-negative bacteria in having adopted an arthropod-borne route of transmission. Y. pestis has evolved in such a way as to be transmitted during the brief encounter between a feeding flea and a host. A transmissible infection primarily depends on the ability of Y. pestis to grow in the flea as a biofilm that is embedded in a complex extracellular matrix. Bacteria in the biofilm phenotype are deposited into the dermis together with flea saliva, elements which cannot be satisfactorily mimicked by needle-injection of Y. pestis from laboratory cultures. The objective of this project is to identify and determine the function of Y. pestis genes that mediate flea-borne transmission and the initial encounter with the host innate immune system at the infection site in the skin. We study the interaction of Y. pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of wild-type or specific Y. pestis mutants. We seek to identify Y. pestis genes that are required for the bacteria to infect the flea midgut and to produce a biofilm that blocks the flea foregut and that is required for efficient transmission. The strategy entails first identifying bacterial genes that are differentially expressed in the flea by gene expression analysis and other techniques. Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors allows further studies into the molecular mechanisms of the bacterial infection of the flea vector. Detailed understanding of the interaction with the insect host may lead to novel strategies to interrupt the transmission cycle. During the last year, we continued to investigate the regulation of Y. pestis genes required for the biofilm life stage in the flea vector. This year we showed that a major bacterial gene regulatory system (known as PhoP-PhoQ) is induced during infection of the flea vector and is required to produce a normal transmissible infection. Because PhoP-PhoQ upregulates several genes required for bacterial resistance to innate immunity, induction of the PhoP-PhoQ system in the arthropod vector prior to transmission may preadapt Y. pestis to resist the initial encounter with the mammalian innate immune response. We also continue to investigate the genetic changes that led to the evolutionarily recent transition of Y. pestis to an arthropod-borne transmission route. Many nonfunctional genes (pseudogenes) occur on the Y. pestis genome; some of them are implicated in regulating the biofilm phenotype that enhances transmission by fleas. We have found that restoration of four pseudogenes with the equivalent functional genes of Yersinia pseudotuberculosis, the recent ancestor of Y. pestis, reduces transmissibility in the flea. Conversely, mutation of these genes in Y. pseudotuberculosis results in the gain of biofilm-forming ability in the flea and loss of oral toxicity to fleas. Thus, gene loss appears to have played a significant role in Y. pestis evolution. In collaboration with the Genomics Unit of the RML Research Technologies Branch, we are examining and characterizing the transcriptomes of Y. pestis, Y. pseudotuberculosis, and our biofilm-producing Y. pseudotuberculosis mutant during growth in vitro and during infection of the flea, with the goal of identifying genes and gene regulatory pathways that are important for flea-borne transmission. The in vivo and in vitro transcriptomic comparisons between these three strains are designed to broadly identify candidate components of biofilm regulatory pathways and other genes important for the recent evolutionary adaptation to flea-borne transmission. Significant differences in the expression of orthologous genes in the flea might be indicative of evolutionary changes in gene regulatory pathways. Moreover, the comparison of Y. pestis and Y. pseudotuberculosis transcripts grown in vitro would help us to select gene candidates involved in the acute toxicity of Y. pseudotuberculosis for fleas. In a separate study published during FY2013, we investigated the possible role of Y. pestis insecticidal-like toxins that are highly expressed during infection of the flea. We found that, although these toxin-like genes are highly induced during Y. pestis infection of the flea, they are neither toxic to fleas nor required to establish a normal transmissible infection. Instead, they appear to have a role after transmission in resisting phagyocytosis by host neutrophils and macrophages. We have also further developed models to examine host-parasite interactions in the dermis after transmission by flea bite, and are completing a project on the host immune response to flea saliva and how this might influence transmission. Last year we established a colony of the ground- and rock-squirrel flea Oropsylla montana, an important vector of plague in the U.S. This year we have done several experimental transmission trials to compare biofilm-independent early-phase and biofilm-dependent transmission mechanisms, and the overall transmission efficiency and the biofilm-dependent blockage ability of Y. pestis in X. cheopis and O. montana fleas.